The brain and thus nerve cells and their function have during the last decades become an increasing subject of scientific investigations. Without doubt, the proper function of this complex system is extremely important for the proper function of the body and mind. It has been found that physical and mental malfunction can be related to i.a. abnormalities in level of signalling compounds, including neurotransmitters. Some malfunctions can be related to decay of nerve cells (neurones), connections between nerve cells and connections between muscle cells and nerve cells. This is e.g. the case in neurodegenerative diseases such as Alzheimer's Disease, where death of nerve cells leads to senility.
During the development of the brain, connections between nerve cells (neurones) are formed. Such connections are necessary for communication between neurones to occur, allowing individual neurones to function together as a whole. In the mature brain, connections between neurones are constantly remodulated to accommodate new demands from a changing environment. The ability to remodulate neural connections is crucial in learning and memory and in regeneration, e.g. after damage to the brain or in neurodegenerative diseases.
It is believed that the mechanisms controlling the formations of neural contacts are generally similar in the developing and the mature. Several mechanisms are involved in the formation of contacts between neurones including cell adhesion, the formation of nerve cell extensions (neurites), fasciculation (bundling of individual neurites) and formation of contact points (synapses).
Cell adhesion molecules (CAMs) constitute a group of proteins mediating adhesion between cells. A major group of CAMs belongs to the immunoglobulin (Ig) superfamily characterised by the presence of immunoglobulin domains. The neural cell adhesion molecule (NCAM) is such a cell adhesion molecule of the Ig superfamily that is particularly abundant in the nervous system. NCAM is expressed in the outer membrane of nerve cells. When one NCAM molecule binds to another NCAM molecule on another cell, the binding between the two cells is strengthened. NCAM not only binds to NCAM but also to other proteins found on nerve cells or in the extracellular substance of the brain (the extracellular matrix). By mediating adhesion between nerve cells—or between nerve cells and the extracellular matrix—NCAM influences migration of cells, extension of neurites, fasciculation of neurites and formation of synapses.
NCAM expression is correlated with morphogenic events suggesting that NCAM is important during development (Edelman 90). Thus, NCAM as believed to be important for the development of the nervous system (Daston et al 1996) and various organs including the kidney (Lackie et al 1990), the liver (Knittel et al 1996), the bowel (Romanska et al 1996), the heart (Gaardsvoll et al 1993), the gonads (Møller et al 1991), the pancreas (Møller et al 1992), and the muscles (Landmesser et al 1990). Therefore, ligands capable of influencing NCAM function may potentially be beneficial in conditions of impaired development of these organs by inducing appropriate differentiation of target cells (Walsh et al 1990). In the brain, the role of NCAM has been supported by knock out mice which have altered development of certain brain regions, including the olfactory system, the hippocampus, the cerebellum and the retina (Cremer et al 1994). In these tissues, the lack of NCAM expression impairs migration of cells (Ono et al 1994) and outgrowth and fasciculation of neurites (Cremer et al 1997) which in turn leads to altered synaptogenesis and morphological and functional changes. Transgenic mice with a change in the NCAM gene to produce only soluble NCAM forms die before birth further indicating that NCAM functions have great potential to interfere with development (Rabinowitz et al 1996).
In the mature nervous system, NCAM have been shown to be important for the plasticity of neuronal connections associated with regeneration, learning and memory (Fields et al 1996). In the peripheral nervous system, NCAM is believed to be necessary for outgrowth of nerve fibres and formation of nerve-muscle connections in regeneration after damage including lesions (Nieke et al 1985) and stroke (Jucker et al 1995).
Moreover, NCAM is presumably involved in ageing-related impairments in the ability to regenerate peripheral nerves and nerve-muscle connections (Olsen et al 1995) as well as in a number of degenerative muscle diseases (Walsh et al 1985). A similar role of NCAM has been observed in the central nervous system where NCAM is believed to be important for neuritic outgrowth, fasciculation, branching and probably target recognition associated with regeneration (Daniloff et al 1986). In addition, NCAM-MAG double knock out mice have shown that NCAM is also necessary for myelination of neuronal fibres which is of crucial importance for neuronal function (Carenini et al 1997). In learning, subtle remodelling of neuronal connections is necessary for the stabilisation of a memory trace and it has been shown that NCAM expression changes concomitant with such changes (Doyle et al 1992). Moreover, interference with NCAM function by antibodies or in knock out mice impairs the ability to learn (Luthi et al., 1994; Rønn et al., 1995; Scholey et al 1993). From knock out mice, it has become evident that NCAM is also involved in other behavioural phenomena. Thus, NCAM knock out mice have altered circadian rhythm (Shen et al 1997) and males shown increased aggression (Stork et al 1997). In humans, elevated levels of soluble NCAM forms have been shown in schizophrenia (van Kammen et al 1998) and sclerosis (Massaro et al 1987) suggesting that NCAM could be of importance for these diseases.
NCAM is found in three main forms of which two are transmembrane forms while the third form is attached to the membrane by a lipid anchor (see FIG. 1) All three forms have the same structure extracellularly consisting of five immunoglobulin domains (Ig domains) and two fibronectin like domains (FnIII domains). A precursor form of the NCAM contains a signal sequence. The amino acid sequence of 140 Kd isoform precursor of human NCAM, is shown in FIG. 17. The Ig domains are numbered one to five from the N-terminal, that is Ig1 to Ig5. The fibronectin domains are likewise called FnIII1 and FnIII2. In addition to mediating cell adhesion, NCAM affect signal transduction in cells (Schuch et al 1989). When an NCAM molecule at the cell surface binds to another cell, a signal is transmitted to the interior of the cell (transmembrane signalling). Within the cell, a signalling cascade is activated that subsequently influences the behaviour of the cell it has been shown that signalling initiated by NCAM binding can stimulate neurite extension (Doherty et al., 1996).
It is unclear, which of the NCAM domains mediate cell adhesion and signal transduction. The generally accepted hypothesis predicts that homophilic NCAM adhesion is mediated by a transreciprocal interaction between the Ig3 domains of two opposing NCAM molecules. Considerable evidence supports this notion and a putative binding site has been identified (Rao et al 1992, Rao et al 1994, Sandig et al 1994). Also ligands affecting the Ig3 domain have been shown to inhibit NCAM mediated cell adhesion. A recent hypothesis predicts that not only the Ig3 but all five Ig-domains mediate homophilic NCAM binding (Ranheim 96). According to this hypothesis, Ig1 of one NCAM molecule binds to Ig5 of another NCAM molecule, Ig2 binds Ig4 and Ig3 binds to Ig3. Thus these two theories of NCAM binding are partially overlapping. The present inventors and their colleagues have recently proposed that a double reciprocal interaction between Ig1 and Ig2 domains of two opposing NCAM molecules may mediate homophilic NCAM binding (Thomsen et al. (1996), Kiselyov et al. (1997), Rønn (1997). Rønn observed an inhibition of aggregation of neurones in a culture of hippocampal cells when adding small peptides which were previously identified as capable of binding to the NCAM Ig1 domain. An additional stimulation of neurite outgrowth was also seen. Rønn neither disclosed the sequence of the peptides studies nor suggested an exploitation of his observations in medical treatment. In conclusion, the mechanism of homophilic NCAM binding is still a matter of debate although most researchers in the field favour the hypothesis of a an reciprocal interaction between all five Ig domains or at least between the Ig3 domains of two opposing NCAM molecules.
Antibodies against NCAM, purified NCAM protein and recombinant NCAM domains have been shown to induce signal transduction in certain cells. High concentrations of NCAM antibody can induce a transient calcium increase as well as a pH change in some but not all neuronal cells. (Schuch et al 1989). The recombinant NCAM domains Ig1 and Ig2 and the combined domains Ig1-5 can induce a similar transient calcium increase and change in pH in certain cells (Frei et al 1992). When used as a substrate or expressed by a monolayer of cells, the NCAM protein can stimulate neurite extension. The response depends on an interaction between the FnIII domains of NCAM with fibroblast growth factor (FGF)-receptors (Doherty et al 1996). In addition, an interaction between the cytoplasmic part of NCAM with the tyrosine kinase fyn is of importance for neurite outgrowth (Beggs et al 1997).
This interaction is believed to activate the Ras-MAP-Kinase pathway (Schmid, R-S et al 1999).
Also, recombinant NCAM domains immobilised to the substratum can stimulate neurite extension, branching of neurites or fasciculation of neurites. Thus the FnIII domains of NCAM can increase branching of neurites when used as a substratum (Stahlhut et al 1997, Kasper et al 1996). Moreover, the FnIII domains have been reported to be the most potent NCAM, domains to influence cell spreading and neurite outgrowth. Ig 1-5 also influenced these processes but less potently than the FnIII domains (Frei et al 1992). In contrast, Ig1 and Ig2 most potently promoted cell adhesion and cell migration in this study (Frei et al 1992). Frei et al also observed stimulation of neurite outgrowth by the isolated NCAM domains Ig3, Ig4, Ig5, FnIII,1 and FnIII,2, but not by Ig1 and Ig2. A sequence located between the Ig5 and the FnIII,1 domains have been shown to be important for fasciculation of neurites (Pollerberg et al 1993). The Ig5 domain, of NCAM is of major importance for neurite outgrowth due to the presence or absence of the sugar chains polysialic acid (PSA) on this domain (Rutishauser et al 1996). Likewise, the Ig4 domain is important due to the presence or absence of the alternatively spliced domain VASE (Doherty et al 1992). Synthetic peptides corresponding to the VASE sequence have been shown to interfere with NCAM stimulated neurite outgrowth (Lahrtz et al 1997). Moreover, the NCAM Ig4 domain is presumed to bind another cell adhesion molecule, L1, and thereby to influence neurite outgrowth (Horstkorte et al 1993). In contrast to the effect of immobilised reagents, NCAM antibodies or recombinant domains inhibit neurite outgrowth when added in solution. Peptides corresponding to the presumed homophilic binding site in Ig3 or mutations in this sequence in the Ig3 domain have been shown to inhibit neurite outgrowth stimulated by NCAM (Sandig et al 1994).
However, an antibody against NCAM has recently been shown to stimulate neurite outgrowth (U.S. Pat. No. 5,667,978). This antibody recognises the Ig3 domain of NCAM. All NCAM domains have moreover been shown to influence proliferation of glial cells, neuroblastoma cells and fibroblasts, the Ig3 domain being the most potent. This function has been shown to require interaction with MAP kinase activity (Krushel 1998). It has been shown that various inhibitory ligands of the NCAM Ig3 domain, including small peptides corresponding to parts of the Ig3 domain sequence, can inhibit glial proliferation (WO 96/18103).
These data suggest, that the NCAM protein or NCAM ligands could potentially influence functions of the nervous system and other tissues. Inhibiting glial proliferation would potentially be beneficial in degenerative conditions (WO 96/18103, U.S. Pat. No. 5,625,040, U.S. Pat. No. 5,667,978). Alternatively, if NCAM functions, particularly the induction of neurite outgrowth, could be stimulated, a beneficial effect on brain function would be possible. A stimulation of certain in vitro NCAM functions has been described for an antibody against NCAM Ig3 (U.S. Pat. No. 5,667,978). However, no small ligands of NCAM with significant stimulatory effect on NCAM functions has been described. Moreover, it is not evident to which NCAM domain such a ligand should be targeted. Most evidence points at the NCAM Ig3 domain as the crucial domain for homophilic binding while the cytoplasmic part of NCAM together with the FnIII domains are presumed to be most important for interactions with signalling molecules.
In the Ph.D. thesis “NCAM and Neural Plasticity” (Rønn 1997), the role of NCAM in neural plasticity was studied. Different assays (test systems), including aggregation of neural cells, neurite extension and long-term potentiation (LTP) were used to study how the role or effect of NCAM was influenced by NCAM antibodies, NCAM fusion proteins and other NCAM ligands. Presumed NCAM ligands selected from a random peptide library were studied. The peptides were found to be able to bind Ig1. One specific peptide, which is not characterised further in the thesis, was shown to inhibit aggregation of neural cells and to stimulate neurite outgrowth. It is concluded that such ligands might be a valuable tool in the continued attempts to clarify the role of NCAM in the developing nervous system as well as in synaptic plasticity. A possible medical use of the investigated peptides is neither an object of the thesis nor suggested therein. Furthermore, the thesis does not disclose the sequences of the investigated peptides.
U.S. Pat. No. 5,625,040 relates to chondroitin sulphate proteoglycan (Phosphacan) and its use in enhancing regeneration of nerves by binding to NCAM. The Phosphacan sequence is 1616 amino acid residues long. Recombinant Phosphacan was obtained by cloning the encoding gene in a suitable vector. The gene was isolated using primers chosen in accordance with the identified amino acid sequences of some proteolytic fragments of Phosphacan. None of the fragments was suggested to possess a biological effect per se.
A stimulatory effect on the potential for neurite extension may be expected to have a beneficial effect in functions of the nervous system requiring plasticity of connections between nerve cells. Such functions include learning and memory and regeneration. It is therefore of considerable interest to identify substances with the capability to influence NCAM mediated signalling.